Abstract
Genetic markers (DNA barcodes) are often used to support and confirm species identification. Barcode sequences can be generated in the field using portable systems based on the Oxford Nanopore Technologies (ONT) MinION sequencer. However, to achieve a broader application, current proof-of-principle workflows for on-site barcoding analysis must be standardized to ensure a reliable and robust performance under suboptimal field conditions without increasing costs. Here, we demonstrate the implementation of a new on-site workflow for DNA extraction, PCR-based barcoding, and the generation of consensus sequences. The portable laboratory features inexpensive instruments that can be carried as hand luggage and uses standard molecular biology protocols and reagents that tolerate adverse environmental conditions. Barcodes are sequenced using MinION technology and analyzed with ONTrack, an original de novo assembly pipeline that requires as few as 1000 reads per sample. ONTrack-derived consensus barcodes have a high accuracy, ranging from 99.8 to 100%, despite the presence of homopolymer runs. The ONTrack pipeline has a user-friendly interface and returns consensus sequences in minutes. The remarkable accuracy and low computational demand of the ONTrack pipeline, together with the inexpensive equipment and simple protocols, make the proposed workflow particularly suitable for tracking species under field conditions.
Highlights
Recent advances in molecular biology allow the use of genetic markers (DNA barcodes) to support and confirm morphological evidence for species identification and to quantify interspecific differences in order to compare species in terms of evolutionary distance
The third-generation sequencer Oxford Nanopore Technologies (ONT, Oxford, UK) MinION has proven successful for sequencing under extreme field conditions, such as the tropical rainforests of Tanzania, Ecuador and Brazil [1,2,3], the hot savannah of West Africa [4], and the ice floes of Antarctica [5]
When we returned to Europe, the same genomic fragments were amplified and sequenced from the same DNA extracts using the Sanger method to evaluate the accuracy of the MinION-based barcoding pipeline
Summary
Recent advances in molecular biology allow the use of genetic markers (DNA barcodes) to support and confirm morphological evidence for species identification and to quantify interspecific differences in order to compare species in terms of evolutionary distance. Most barcodes are still generated using the Sanger sequencing method, which requires access to a well-equipped molecular biology laboratory. Several groups have reported successful on-site barcoding, it remains difficult to perform molecular biology procedures in sub-optimal and extreme environments. The quality of sequences generated in the field was consistently lower than that achieved in the laboratory, suggesting that reagents and flow cells were affected by the unstable shipping and/or environmental conditions [1]. The identification of robust protocols and equipment that tolerates suboptimal transport and operating conditions (but remains simple, inexpensive, and portable) is highly desirable in order to exploit the full potential of barcode sequencing in the field
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